scholarly journals Yersinia pestis Targets the Host Endosome Recycling Pathway during the Biogenesis of the Yersinia-Containing Vacuole To Avoid Killing by Macrophages

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Michael G. Connor ◽  
Amanda R. Pulsifer ◽  
Donghoon Chung ◽  
Eric C. Rouchka ◽  
Brian K. Ceresa ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Host Factors ◽  
Endocytic Recycling ◽  
Recycling Endosome ◽  
Independent Manner ◽  
Genome Wide ◽  
Type Three Secretion System ◽  
A Genome ◽  
Recycling Pathway ◽  
Bacterial Replication

ABSTRACTYersinia pestishas evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis,Y. pestisprevents phagolysosome maturation and establishes a modified compartment termed theYersinia-containing vacuole (YCV).Y. pestisactively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requiresY. pestisinteractions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required forY. pestissurvival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival ofY. pestis. Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated thatY. pestisactively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV byY. pestisto resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered thatY. pestisdisrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence thatY. pestistargets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV.IMPORTANCEYersinia pestiscan infect and survive within macrophages. However, the mechanisms that the bacterium use to subvert killing by these phagocytes have not been defined. To provide a better understanding of these mechanisms, we used an RNAi approach to identify host factors required for intracellularY. pestissurvival. This approach revealed that the host endocytic recycling pathway is essential forY. pestisto avoid clearance by the macrophage. We further demonstrate thatY. pestisremodels the phagosome to resemble a recycling endosome, allowing the bacterium to avoid the normal phagolysosomal maturation pathway. Moreover, we show that infection withY. pestisdisrupts normal recycling in the macrophage and that disruption is required for bacterial replication. These findings provide the first evidence thatY. pestistargets the host endocytic recycling pathway in order to evade killing by macrophages.

scholarly journals A genome-wide CRISPR screen identifies UFMylation and TRAMP-like complexes as host factors required for hepatitis A virus infection

Cell Reports ◽  
2021 ◽  
Vol 34 (11) ◽  
pp. 108859
Author(s):  
Jessie Kulsuptrakul ◽  
Ruofan Wang ◽  
Nathan L. Meyers ◽  
Melanie Ott ◽  
Andreas S. Puschnik
Keyword(s):  
Virus Infection ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Host Factors ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

scholarly journals A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets

PLoS Biology ◽  
2021 ◽  
Vol 19 (12) ◽  
pp. e3001490
Author(s):  
Annika Kratzel ◽  
Jenna N. Kelly ◽  
Philip V’kovski ◽  
Jasmine Portmann ◽  
Yannick Brüggemann ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Huh7 Cell ◽  
Host Factors ◽  
Genome Wide ◽  
A Genome ◽  
Dependent Inhibition ◽  
Crispr Screen ◽  
Human Coronaviruses ◽  
Approved Drugs ◽  
Huh7 Cell Line

Over the past 20 years, 3 highly pathogenic human coronaviruses (HCoVs) have emerged—Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and, most recently, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)—demonstrating that coronaviruses (CoVs) pose a serious threat to human health and highlighting the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycles. Herein, we conducted 2 independent genome-wide CRISPR/Cas-9 knockout (KO) screens to identify MERS-CoV and HCoV-229E host dependency factors (HDFs) required for HCoV replication in the human Huh7 cell line. Top scoring genes were further validated and assessed in the context of MERS-CoV and HCoV-229E infection as well as SARS-CoV and SARS-CoV-2 infection. Strikingly, we found that several autophagy-related genes, including TMEM41B, MINAR1, and the immunophilin FKBP8, were common host factors required for pan-CoV replication. Importantly, inhibition of the immunophilin protein family with the compounds cyclosporine A, and the nonimmunosuppressive derivative alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures, which recapitulate the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrated that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

scholarly journals A genome-wide CRISPR screen identifies interactors of the autophagy pathway as conserved coronavirus targets

2021 ◽  
Author(s):  
Annika Kratzel ◽  
Jenna N. Kelly ◽  
Yannick Brueggemann ◽  
Jasmine Portmann ◽  
Philip V’kovski ◽  
...  
Keyword(s):  
Host Factors ◽  
The Novel ◽  
Genome Wide ◽  
A Genome ◽  
Dependent Inhibition ◽  
Crispr Screen ◽  
Dose Dependent Inhibition ◽  
Autophagy Pathway ◽  
Approved Drugs ◽  
Dose Dependent

SummaryOver the past 20 years, the emergence of three highly pathogenic coronaviruses (CoV) – SARS-CoV, MERS-CoV, and most recently SARS-CoV-2 – has shown that CoVs pose a serious risk to human health and highlighted the importance of developing effective therapies against them. Similar to other viruses, CoVs are dependent on host factors for their survival and replication. We hypothesized that evolutionarily distinct CoVs may exploit similar host factors and pathways to support their replication cycle. Here, we conducted two independent genome-wide CRISPR/Cas9 knockout screens to identify pan-CoV host factors required for the replication of both endemic and emerging CoVs, including the novel CoV SARS-CoV-2. Strikingly, we found that several autophagy-related genes, including the immunophilin FKBP8, TMEM41B, and MINAR1, were common host factors required for CoV replication. Importantly, inhibition of the immunophilin family with the compounds Tacrolimus, Cyclosporin A, and the non-immunosuppressive derivative Alisporivir, resulted in dose-dependent inhibition of CoV replication in primary human nasal epithelial cell cultures that resemble the natural site of virus replication. Overall, we identified host factors that are crucial for CoV replication and demonstrate that these factors constitute potential targets for therapeutic intervention by clinically approved drugs.

scholarly journals A Genome-Wide siRNA Screen to Identify Host Factors Necessary for Growth of the Parasite Toxoplasma gondii

PLoS ONE ◽  
2013 ◽  
Vol 8 (6) ◽  
pp. e68129 ◽  
Author(s):  
Lindsey A. Moser ◽  
Angela M. Pollard ◽  
Laura J. Knoll
Keyword(s):  
Toxoplasma Gondii ◽  
Host Factors ◽  
Sirna Screen ◽  
Genome Wide ◽  
A Genome

scholarly journals A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection

2019 ◽  
Vol 93 (13) ◽  
Author(s):  
Rutger D. Luteijn ◽  
Ferdy van Diemen ◽  
Vincent A. Blomen ◽  
Ingrid G. J. Boer ◽  
Saravanan Manikam Sadasivam ◽  
...  
Keyword(s):  
Virus Infection ◽  
Vaccinia Virus ◽  
Heparan Sulfate ◽  
Host Cell ◽  
Host Factors ◽  
Virus Infectivity ◽  
Sequencing Analysis ◽  
Vaccinia Virus Infection ◽  
Genome Wide ◽  
A Genome

ABSTRACTVaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCEPoxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.

scholarly journals Uncovering Flavivirus Host Dependency Factors through a Genome-Wide Gain-of-Function Screen

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 68 ◽  
Author(s):  
Evgeniya Petrova ◽  
Ségolène Gracias ◽  
Guillaume Beauclair ◽  
Frédéric Tangy ◽  
Nolwenn Jouvenet
Keyword(s):  
Ribosomal Protein ◽  
Host Factors ◽  
Global Public Health ◽  
Host Proteins ◽  
Gain Of Function ◽  
Genome Wide ◽  
Human Genes ◽  
A Genome ◽  
Ribosomal Protein S3

Flaviviruses, such as dengue (DENV), West Nile (WNV), yellow fever (YFV) and Zika (ZIKV) viruses, are mosquito-borne pathogens that present a major risk to global public health. To identify host factors that promote flavivirus replication, we performed a genome-wide gain-of-function cDNA screen for human genes that enhance the replication of flavivirus reporter particles in human cells. The screen recovered seventeen potential host proteins that promote viral replication, including the previously known dolichyl-diphosphooligosaccharide--protein glycosyltransferase non-catalytic subunit (DDOST). Using silencing approaches, we validated the role of four candidates in YFV and WNV replication: ribosomal protein L19 (RPL19), ribosomal protein S3 (RPS3), DDOST and importin 9 (IPO9). Applying a panel of virological, biochemical and microscopic methods, we validated further the role of RPL19 and DDOST as host factors required for optimal replication of YFV, WNV and ZIKV. The genome-wide gain-of-function screen is thus a valid approach to advance our understanding of flavivirus replication.

scholarly journals Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Bo Li ◽  
Sara M. Clohisey ◽  
Bing Shao Chia ◽  
Bo Wang ◽  
Ang Cui ◽  
...  
Keyword(s):  
Virus Infection ◽  
Influenza A Virus ◽  
Influenza A ◽  
Meta Analysis ◽  
Host Factors ◽  
Validation Data ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Influenza A Virus Infection

AbstractHost dependency factors that are required for influenza A virus infection may serve as therapeutic targets as the virus is less likely to bypass them under drug-mediated selection pressure. Previous attempts to identify host factors have produced largely divergent results, with few overlapping hits across different studies. Here, we perform a genome-wide CRISPR/Cas9 screen and devise a new approach, meta-analysis by information content (MAIC) to systematically combine our results with prior evidence for influenza host factors. MAIC out-performs other meta-analysis methods when using our CRISPR screen as validation data. We validate the host factors, WDR7, CCDC115 and TMEM199, demonstrating that these genes are essential for viral entry and regulation of V-type ATPase assembly. We also find that CMTR1, a human mRNA cap methyltransferase, is required for efficient viral cap snatching and regulation of a cell autonomous immune response, and provides synergistic protection with the influenza endonuclease inhibitor Xofluza.

scholarly journals Genome-wide CRISPR-dCas9 screens inE. coliidentify essential genes and phage host factors

2018 ◽  
Author(s):  
François Rousset ◽  
Lun Cui ◽  
Elise Siouve ◽  
Florence Depardieu ◽  
David Bikard
Keyword(s):  
High Throughput ◽  
Essential Genes ◽  
Host Factors ◽  
Null Mutant ◽  
Genetic Screens ◽  
E Coli ◽  
Colanic Acid ◽  
Genome Wide ◽  
A Genome

AbstractHigh-throughput genetic screens are powerful methods to identify genes linked to a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide CRISPR-dCas9 screen using a pool of ~ 92,000 sgRNAs which target random positions in the chromosome ofE. coli. We first investigate the utility of this method for the prediction of essential genes and various unusual features in the genome ofE. coli. We then apply the screen to discoverE. coligenes required by phages λ, T4 and 186 to kill their host. In particular, we show that colanic acid capsule is a barrier to all three phages. Finally, cloning the library on a plasmid that can be packaged by λ enables to identify genes required for the formation of functional λ capsids. This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria in order to identify genes linked to a given phenotype.

scholarly journals Genome-wide CRISPR activation screen identifies novel receptors for SARS-CoV-2 entry

2021 ◽  
Author(s):  
Shiyou Zhu ◽  
Ying Liu ◽  
Zhuo Zhou ◽  
Zhiying Zhang ◽  
Xia Xiao ◽  
...  
Keyword(s):  
Virus Entry ◽  
Host Factors ◽  
The Novel ◽  
Host Proteins ◽  
Alternative Pathways ◽  
Genome Wide ◽  
A Genome ◽  
Novel Host ◽  
Shed Light ◽  
Crispr Activation

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been endangering worldwide public health and economy. SARS-CoV-2 infects a variety of tissues where the known receptor ACE2 is low or almost absent, suggesting the existence of alternative pathways for virus entry. Here, we performed a genome-wide barcoded-CRISPRa screen to identify novel host factors that enable SARS-CoV-2 infection. In addition to known host proteins, i.e. ACE2, TMPRSS2, and NRP1, we identified multiple host components, among which LDLRAD3, TMEM30A, and CLEC4G were confirmed as functional receptors for SARS-CoV-2. All these membrane proteins bind directly to spike's N-terminal domain (NTD). Their essential and physiological roles have all been confirmed in either neuron or liver cells. In particular, LDLRAD3 and CLEC4G mediate SARS-CoV-2 entry and infection in a fashion independent of ACE2. The identification of the novel receptors and entry mechanisms could advance our understanding of the multiorgan tropism of SARS-CoV-2, and may shed light on the development of the therapeutic countermeasures against COVID-19.

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